Mechanism and Substrate Specificity of tRNA–Guanine Transglycosylases (TGTs): tRNA‐Modifying Enzymes from the Three Different Kingdoms of Life Share a Common Catalytic Mechanism

Abstract

Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.

Novel genomic island modifies DNA with 7-deazaguanine derivatives

“…2B). The key residues that catalyze the G exchange (Asp102 and Asp280 of Zymomonas mobilis bTGT and Asp95 and Asp249 of Pyrococcus horikoshii aTGT) (19), as well as the Zinc binding site (CXCXXCX 22 H motif), are conserved in TgtA5 (Fig. 2B).…”

“…Analysis of the substrate binding pocket suggests that TgtA5 binds preQ 0 , like the aTGTs. The critical residues for preQ 0 binding by aTGT are GVVPL[LM] at positions 196-201 of the P. horikoshii enzyme, differing from the bTGT preQ 1 binding pocket residues GLAVGE at position 230-235 of the Z. mobilis enzyme (19). Alignments of TgtA5 sequences with bTGT and aTGT showed the binding pocket residues resembled aTGT more than bTGT (G[ML]VPL[KR] in (Fig.…”

“…The TgtA5 domain of these proteins is C1 and C2 represent C-terminal domains unique to aTGT (17,19). (B) Model and alignments of proposed substrate-binding pocket of TgtA5.…”

“…1A). Although the bTGT and aTGT recognize a guanosine at different positions of the tRNA and use different substrates, key residues involved in base exchange and in zinc binding are conserved (19) (Fig. 2A).…”

“…2A). In addition, signature residues involved in the differences in 7-deazaguanosine substrate recognition between the bTGT and aTGT enzymes have been identified (19) (Fig. 2B).…”